The ontogeny of the human globin genes is an important focus of study both with respect to the fundamental developmental biology and molecular genetics of the control of expression of this complex gene system, but also because modifying this developmental control would be of therapeutic value in the treatment of the prevalent genetic diseases of hemoglobin. We are studying this problem from several aspects. First, we have improved culture methods for human erythroid precursors, by antibody selection, so as to obtain purified populations of these cells to establish at which developmental stage drugs change their phenotype. Second, we have developed real-time, quantitative PCR methods to measure globin gene expression in single cells and have used these methods to analyze the mechanisms of the effects of chemically useful drugs, such as hydroxyurea and butyrate, on globin gene expression. Third, we have recently shown that nitric oxide donors, such as cysteine-NO, can induce fetal hemoglobin expression in K562 cells as well as the purified erythroid (CD34) precursor cells described above. Induction of fetal hemoglobin was demonstrated both by quantitative PCR of gamma-globin mRNA and by HPLC of fetal hemoglobin protein. Further, we have shown that both this agent and hydroxyurea acts by stimulating guanylyl cyclase and increasing cyclic GMP levels; conversely, inhibitors of guanylyl cyclase block the action of both agents. In more recent studies we have shown that hydroxyurea induces NO production in macrophages and endothelial cells-in part through inhibition of proteasome degradation of eNOS protein-and that this efect may allow a mechanism for a paracrine efect of NO on erythroid progenitors from these signalling molecules produceed by stromal and other bone marrow cells. In current studies of globin production-at the mRNA and protein levels-we are studying the mechansims of such singalling in hemoglobin ontogeny and its therapeutic implications. These results raise the possiblity that agents which affect this cyclic nucleotide pathway, or other signalling pathways, can be used to increase fetal hemoglobin levels in patients with sickle cell anemia and thalassemia. Such therapeutic advances have been the focus of this research program for almost three decades. In recent studies we have been analyzing the change in fetal hemoglobin levels in normal newborns with age and fine-using both quantitative protein and mRNA measurements-that there appear to be two distinct silencing mechansims for fetal hemoglobin-one primarily cellular and one primarily with respect to transcription mechanisms. We are now studying these processes in sickle cell children and will examine the effects of drugs on these silencing mechansims.